Sven Falk

It has been broadly recognized that the crosstalk between cells and their extracellular matrix (ECM) is crucial for the proper function of biological tissues. Relatively recently the role of ECM came in focus in the context of neuronal development and regeneration, where the effects of the ECM mechanics on the migration of neurons and neurite growth are still incompletely understood. Here we present an in silico twin framework for neurite growth focusing on its biophysical interactions with the ECM. This coarsegrained model accounts for viscoelastic liquid- and solid-like ECMs and neurite growth by ECM-mediated traction forces. Resulting growth trajectories can be rationalized based on the theory of random walks and polymer physics. To critically assess model’s predictive power, we performed experiments on neurites of hippocampal rat neurons growing in 3D collagen gels and observed a more persistent axon outgrowth in denser matricies. The model fully recapitulated the effect, thereby underpinning the central role of mechanical interactions with ECM as guiding principle of axonal growth. We argue that a combination our model with optical microscopy may provide an is silico twin helping to disentangle the contributions of “passive” physics from more complex effects of chemical queues or an apparent mechanosensing.

During neurodevelopment neural stem cells give rise to a spatially patterned tissue in which a regionally differentially regulated balance between proliferation and differentiation produces the fine-tuned number of neurons and macroglia necessary for a functional central nervous system. The cells driving these highly intricated developmental processes of patterning, growth and differentiation are constantly exposed to a mechanical environment that is, however, variable between different brain regions and along differentiation trajectories. Here we demonstrate that both, acute mechanical manipulations as well as a persistent change in the mechanical environment provided to human brain organoids, instruct neural stem cell lineage decisions. Furthermore, we dissect the underlying changes in the molecular program of organoid-resident cells by bulk- and single cell RNA-sequencing. These data reveal that mechanical manipulations impact on molecular programs governing early patterning events as well as cell-type-specific cellular metabolism. Thus, our results unravel a regulatory network linking mechanics and neural stem cell lineage decisions.

While random X-chromosome inactivation in female cells of placental mammals silences one allele of the majority of X-chromosomal genes, a considerable fraction is only incompletely and variably inactivated. Human model systems to study the dynamics of incomplete X-inactivation are limited mostly to postmortem tissue, thereby disregarding developmental trajectories. Here, we used clonal human female induced pluripotent stem cells to track allele-specific expression of X-chromosomal genes along neural differentiation. We discovered dynamic reactivation and late-silencing of gene expression from the inactive X-chromosome leading to differentiation-induced locus- and lineage-specific usage of the two X-chromosomal alleles. In brain organoids modeling Opitz BBB/G syndrome, an X-linked neurodevelopmental disorder, reactivation of alleles from the inactive X-chromosome rescued cellular phenotypes and led to intermediate manifestations in female tissue. Taken together, our data demonstrate that alleles on the inactive X-chromosome can serve as a critical reservoir dynamically used during differentiation, thereby enhancing resilience of female neural tissue.

Summary: Establishing long-range connections during human brain development is an intricate multi-step process disturbed in many neurodevelopmental disorders (NDDs). The aberrant formation of these connections is caused by mutations in a plethora of different genes with distinct molecular functions, triggering the question of whether there are common key downstream mediators at which different pathologies are converging. We employed brain organoids to model early human brain developmental aspects of Coffin-Siris-like 9, Opitz BBB/G, and Pitt-Hopkins syndromes. These NDDs are caused by mutations in SOX11 , MID1 , and TCF4 respectively, and are characterized by a multitude of distinct symptoms yet share alterations in long-range projections as a common feature. Here, we uncover that mutations in all three genes phenotypically converge, showing impaired neurite extension with increased tortuosity and decreased growth speed resulting in shorter beelines. Moreover, the mutant neurites exhibit a decrease in growth persistence providing a conceptual framework explaining why long- but not short-range connections are affected. Correlating with the converging cellular phenotype, molecular characterization revealed a striking convergence on signaling pathways implicated in the interaction of neurites with their extracellular environment. In-silico modeling and perturbation of neurite outgrowth suggest that altered neurite-extracellular environment interactions are sufficient to recapitulate the mutant phenotypes but also facilitate the prediction of specific parameters causing disturbed neurite growth in mutant neurons.

Heterozygous mutations ofTCF4in humans cause Pitt–Hopkins syndrome, a neurodevelopmental disease associated with intellectual disability and brain malformations. Although most studies focus on the role of TCF4 in neural stem cells and neurons, we here set out to assess the implication of TCF4 for oligodendroglial differentiation. We discovered that both monoallelic and biallelic mutations inTCF4result in a diminished capacity to differentiate human neural progenitor cells toward myelinating oligodendrocytes through the forced expression of the transcription factors SOX10, OLIG2, and NKX6.2. Using this experimental strategy, we established a novel organoid model, which generates oligodendroglial cells within a human neurogenic tissue–like context. Also, here we found a reduced ability of TCF4 heterozygous cells to differentiate toward oligodendroglial cells. In sum, we establish a role of human TCF4 in oligodendrocyte differentiation and provide a model system, which allows to dissect the disease etiology in a human tissue–like context.

During neurodevelopment neural stem cells give rise to a spatially patterned tissue in which a regionally differentially regulated balance between proliferation and differentiation produces the fine-tuned number of neurons and macroglia necessary for a functional central nervous system. The cells driving these highly intricated developmental processes of patterning, growth and differentiation are constantly exposed to a mechanical environment that is however variable between different brain regions and along differentiation trajectories. Here we demonstrate that both, acute mechanical manipulations as well as a persistent change in the mechanical environment provided to human brain organoids, instruct neural stem cell lineage decisions. Furthermore, we dissect the underlying changes in the molecular program of organoid-resident cells by bulk- and single cell RNA-sequencing and reveal that mechanical manipulations impact on molecular programs governing early patterning events as well as cell-type specifically alter cellular metabolism. Thus, our results unravel a regulatory network linking mechanics and neural stem cell lineage decisions.

The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked geneMID1. Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of theMID1gene coding for the N-terminal RING domain.

Summary: While random X-chromosome inactivation in female cells of placental mammalians silences one allele of the majority of X-chromosomal genes, a considerable fraction is only incompletely and variably inactivated resulting in a tissue-specific pattern of mono- and biallelic expression. Here we used clonal human female induced pluripotent stem cells (iPSCs) allowing to trace the (in)activation status of the two X-chromosomes individually along neural differentiation trajectories. We discovered X-chromosome-wide locus- and lineage-specific dynamic usage of the two X-chromosomal alleles in female cells induced by differentiation. Leveraging iPSCs derived from patients with an X-linked neurodevelopmental disorder, we demonstrate that activation of alleles on the inactive X-chromosome can exert protective effects on the manifestation of disease phenotypes in female neural cells and tissue. Taken together, our data demonstrate that alleles on the inactive X-chromosome can serve as a critical reservoir reactivated during differentiation, thereby enhancing resilience of female neural tissue.

Most adult hippocampal neural stem cells (NSCs) remain quiescent, with only a minor portion undergoing active proliferation and neurogenesis. The molecular mechanisms that trigger the transition from quiescence to activation are still poorly understood. Here, we found the activity of the transcriptional co‐activator Yap1 to be enriched in active NSCs. Genetic deletion of Yap1 led to a significant reduction in the relative proportion of active NSCs, supporting a physiological role of Yap1 in regulating the transition from quiescence to activation. Overexpression of wild‐type Yap1 in adult NSCs did not induce NSC activation, suggesting tight upstream control mechanisms, but overexpression of a gain‐of‐function mutant (Yap1‐5SA) elicited cell cycle entry in NSCs and hilar astrocytes. Consistent with a role of Yap1 in NSC activation, single cell RNA sequencing revealed a partial induction of an activated NSC gene expression program. Furthermore, Yap1‐5SA expression also induced expression of Taz and other key components of the Yap/Taz regulon that were previously identified in glioblastoma stem cell‐like cells. Consequently, dysregulated Yap1 activity led to repression of hippocampal neurogenesis, aberrant cell differentiation, and partial acquisition of a glioblastoma stem cell‐like signature.

Neuronal heterogeneity has been established as a pillar of higher central nervous system function, but glial heterogeneity and its implications for neural circuit function are poorly understood. Here we show that the adult mouse dentate gyrus (DG) of the hippocampus is populated by molecularly distinct astrocyte subtypes that are associated with distinct DG layers. Astrocytes localized to different DG compartments also exhibit subtype-specific morphologies. Physiologically, astrocytes in upper DG layers form large syncytia, while those in lower DG compartments form smaller networks. Astrocyte subtypes differentially express glutamate transporters, which is associated with different amplitudes of glutamate transporter-mediated currents. Key molecular and morphological features of astrocyte diversity in the mice DG are conserved in humans. This adds another layer of complexity to our understanding of brain network composition and function, which will be crucial for further studies on astrocytes in health and disease.